The broad goal of this proposal is to characterize novel protein interactions involving the inositol trisphosphate receptor (InsP3R) Ca2+ release channel. The InsP3R participates in generation of complex Ca2+ signals that regulate many physiological processes. Interactions between the InsP3R and cellular components could provide effective means to couple Ca2+ release to specific targets, or to regulate Ca2+ release properties of the channel. Recently, we identified a novel mechanism that directly links the InsP3R to programmed cell death. We identified BC!-XL, a pro-survival member of the Bcl-2 family of pro- and anti-apoptotic proteins, as an interactor with the InsP3R. Bel-XL binding sensitizes channel gating to extremely low InsP3 concentrations that exist in unstimulated cells, reducing Ca2+ in the endoplasmic reticulum (ER) and enhancing Ca2+ signaling. The interaction profoundly enhances the ability of cells to withstand apoptotic insults, thereby identifying a molecular mechanism that links the ER, InsP3R and Bcl-2 proteins to cell survival in a novel paradigm. Because apoptosis is orchestrated by Bcl-2 proteins in normal physiology and pathophysiology, our identification of a novel physiological target of Bcl-2 proteins may provide new opportunities for interventions in human diseases involving apoptosis, including cancer, neurodegeneration and cardiovascular disease. We propose three aims to characterize the mechanisms and function of the interaction between the InsP3R and Bcl-2 proteins. First, we will define the mechanisms of Bcl-Xt regulation of the channel using a combination of biochemistry, cell imaging, single channel recording of recombinant InsP3R in native ER membranes, and physiological assays. Second, we will determine the structural bases for the interaction, using mutagenesis, peptide competition and biophysical approaches. We will also examine directly the solution structure of the InsP3R/Bcl-2 complex by NMR spectroscopy. Third, we will determine the physiological relevance of the interaction of pro-survival Bcl-2 proteins with the InsP3R. First, we will identify the mechanisms whereby this interaction confers apoptosis resistance, by testing the hypothesis that mitochondrial bioenergetics is a primary sensor. Second, we will determine if disruption of the interaction affects apoptosis resistance in vivo. The results of these studies should provide important insights into the molecular mechanisms that regulate apoptosis, and into the development of novel targets for therapeutic interventions in human diseases.